ABSTRACT The consensus view of tight junction structure is strands are composed of claudin proteins. Some claudins are thought to form pores that allow paracellular flux while others form the seal that defines the epithelial barrier. An alternative view is that claudins form pores, but not the seal. In this model sealing claudins, i.e. those that enhance barrier function, play a regulatory role by competitively interfering with the function of pore-forming claudins. Neither model has been definitively demonstrated, which limits our ability to develop means to modulate claudin function therapeutically. It is, however, known that claudin proteins interact in both cis, i.e. within a single cell, and trans, i.e. between proteins on adjacent cells, configurations. Further, some progress has been made in defining which claudins are able to interact homo- or heterotypically. These interactions are dynamic, as tight junction protein complexes undergo continuous molecular remodeling at steady state. Specific trans-interactions between claudin extracellular domains have been implicated in both claudin anchoring and strand assembly. While the contribution of cis-interactions to strand assembly is less well defined, it is clear that disruption of claudin-2 anchoring can result in loss of claudin-2 pore function. There are, however, many gaps in these data, including detailed analysis of claudin barrier and pore properties as functions of cis- and trans-interactions. Such information would help to better understand the mechanisms of claudin function. The overall objective of this application is to define claudin protein interactions and determine how these affect claudin pore assembly and function as well as barrier development and maintenance. Such data would fill the knowledge gap, relate integration of claudin functions, and overcome a major obstacle to progress in tight junction biology. My central hypothesis is that claudin-2, a pore-forming claudin, is competitively regulated by claudin-4, a sealing claudin, via disruption of claudin-2 pores. These studies build upon both published reports and my preliminary data showing that claudin-2 and claudin-4 have distinct dynamic behaviors and differentially regulate paracellular permeability of model epithelial monolayers. My hypothesis will be tested by determining the roles of cis- and trans- interactions in claudin-2 anchoring and function and the regulation of these properties by claudin-4 (Aim 1) and by characterizing claudin protein interactions biochemically and in complex multicellular models (Aim 2). The rationale is that improved understanding of claudin pore and sealing functions will make it possible to develop pharmacologic agents that modulate barrier function. While both models of claudin function have merit, I anticipate that my studies will validate the claudin competition model of tight junction. This proposal is innovative because the novel model investigated will provide new perspective on how claudins define paracellular permeability and barrier function. The significance of this proposal is it will move tight junction biology forward and benefit human health by allowing for the creation of more targeted therapeutic treatments for human intestinal barrier dysfunction.